Development of a PCR assay for detection of the thin, binucleate Rhizoctonia causing Eradu patch disease of lupin and barley

摘要

The field symptoms of Eradu patch are distinct, stunted patches in narrow-leaf lupin and ill-thrift patches in barley. The pathogen, a thin, binucleate Rhizoctonia (TBR), is difficult to isolate with standard methods. To develop an assay specific to TBR, the ribosomal RNA ITS region was amplified and a 610 bp section including the conserved 5.8S region was sequenced. This sequence did not match any published sequence. Primers specific to TBR were constructed. The specificity of the assay was confirmed using 104 isolates of TBR obtained from many sites in Western Australia. Tests against R. solani AG 8 and other fungi commonly isolated from lupin and barley roots were negative. TBR was detected in all roots from artificially infected lupin and barley, but detection of TBR in root samples collected from field patches in 1997 was low ( 0 - 15%). New methods for extracting the TBR DNA, including the use of soil immersion plates, enabled detection of TBR ( 80 - 100%) in field samples collected in 1999. The PCR assay developed is a reliable technique for the detection of TBR in field samples and will provide an effective tool for diagnostics and the conduct of field surveys.